山东德州市鸡源隐孢子虫种类的分子鉴定

根据禽源隐孢子虫GP20基因,设计1对保守引物和1对特异性引物,建立巢氏PCR检测方法,并进行验证.该巢氏PCR能特异性地扩增出496bp的禽源隐孢子虫基因组DNA中相应片段,最低能检测到0.52fg的阳性参照DNA.建立的巢氏PCR方法具有高度的特异性和敏感性,可以用于样品的检测.通过该方法鉴定山东省德州地区养鸡场中感染鸡的隐孢子虫种类为C.baileyi,感染率为31.5%.     

实验动物金黄色葡萄球菌PCR检测方法的建立和应用

为了快速检测出实验动物临床样品中的金黄色葡萄球菌（SA），建立了一种普通PCR方法。同时合成3对引物，用金黄色葡萄球菌的阳性核酸DNA做模板，进行引物扩增，筛选出一对能扩增出270 bp单一目的片段的引物。逐一对该引物灵敏度、重复性和特异性进行了测试。实验结果显示，该方法具有高度的特异性，除了识别金黄色葡萄球菌基因组外，对小鼠其他常见病原菌均不发生交叉反应，具有优良的敏感性和稳定性。两个不同操作人员，在两个不同时间段，利用两台不同品牌的PCR仪器，用金黄色葡萄球菌阳性样品进行测试，结果显示此方法再现性良好。用该方法检测待测样本，效果也良好。     

双引物PCR检测抗草甘膦豆粕饲料中的转基因成分

在一个PCR反应体系内,同步扩增大豆内参照基因lec和抗草甘膦大豆外源基因盒中的CaMV35s启动子、nos终止子或cp4 epsps基因,建立转基因大豆双引物PCR检测方法.PCR产物用限制性内切酶酶切进一步鉴定.结果表明,用双引物PCR扩增出标准转基因大豆阳性对照、阿根廷大豆、美国RR大豆及豆粕中的lec基因和相应的特异性外源基因;酶切反应确证PCR产物为特定片段大小的目的序列.建立的双引物PCR法适用于抗草甘膦大豆原料和豆粕饲料中转基因成分的简单化、高精度和高灵敏检测.     

快速诊断小鹅瘟PCR方法的优化建立

为建立快速诊断小鹅瘟的PCR方法,根据已发表的GPV主要结构蛋白VP3基因序列,经多序列比对和Oligo6．0软件分析设计特异性PCR引物。以微量法提取病鹅肝组织DNA,优化PCR反应引物退火温度、循环次数和鉴定其特异性,并与酚氯仿法和煮沸法进行同步对比研究。结果显示,建立的PCR检测方法能够特异性检测病鹅肝组织中GPV的核酸,其扩增片段大小为343bp,最佳退火温度为58℃,最佳循环次数为35次。微量法提取的病毒核酸PCR检测灵敏度分别是酚氯仿法的50倍和煮沸法的2500倍。该PCR检测方法不与鹅副黏病毒、禽流感病毒、传染性支气管炎病毒、鸭瘟病毒以及健康鹅肝组织发生交叉反应。本研究建立的GPVPCR检测方法能够快速诊断小鹅瘟。     

FTA滤膜用于PCR检测豆制品中蜡样芽孢杆菌的研究

采用FTA滤膜从豆制品中直接提取模板DNA,根据蜡样芽孢杆菌的hblA基因设计一对特异性引物进行PcR,并对反应条件进行优化．结果表明该引物能特异性扩增出338bp的目的条带;不增菌的情况下,PCR检测蜡样芽孢杆菌的灵敏度是102CFU／mL,检测时间为4h．     

FTA滤膜用于PCR检测肉中志贺氏菌的研究

建立直接从肉中检测志贺氏菌的快速、敏感、特异的PCR检测方法．根据GenBank公布的志贺氏菌属的侵袭性质粒抗原基因IpaH的保守序列设计特异性引物．经过PCR扩增得到393bp的产物．经过DNA测序证实该产物为目的扩增产物．使用FTA滤膜处理样品．再通过PCR方法检测志贺氏菌．猪肉、牛肉、羊肉检测的检出限均为10CFU／mL匀浆,可在6h内完成对肉中志贺氏菌的检测．FTA滤膜用于PCR检测肉中志贺氏菌灵敏度高,耗时短,为肉中志贺氏菌的快速检测提供了新的方法．     

哈密瓜SSR体系的建立及反应条件的优化

以新疆哈密瓜为试验材料,利用正交试验设计研究了4个主要因子(Mg2+、DNA聚合酶、SSR引物和dNTP)对SSR扩增结果的影响,并对该哈密瓜SSR反应体系进行了优化,同时尝试用降落(Touchdown,TD)PCR方法扩增目的片段.最终筛选出如下最佳反应体系:总反应体积为25 μL,其中dNTP 200 μmol/L,SSR引物0.75μmol/L,Taq DNA聚合酶1.0 U,Mg2+ 1.5 mmol/L;降落PCR省却了常规PCR中摸索最适退火温度的过程,对一些Tm值相近的PCR反应可应用一套温度程序扩增,是一种行之有效的方法.     

荧光定量PCR对微小RNA的检测及应用

目的:建立一种定量、简单、快速、敏感的微小RNA(miRNA,miR)检测方法,并用其观察一些病理状态下有关miRNA的变化.方法:通过特殊设计的茎环结构的反转录引物,对miRNA进行反转录,然后用相应miRNA的正反向引物对该miRNA进行定量检测,观察重复性和特异性.用此法检测了人巨细胞病毒(HCMV)对滋养细胞(HPT-8)细胞内miR-513表达水平的影响,也检测了结直肠癌组织miR-145水平.结果:用该法扩增miRNA特异性强,结果重复性好,该法检测到HCMV感染对miR-513表达的梯度效应.也发现癌组织和正常组织间miR-145的表达有明显的差异.结论:用荧光定量PCR可以对微小RNA快速有效的检测,在临床工作中可得到很好的应用.     

DNA条形码技术在牛肉及其制品鉴别中的应用

利用DNA条形码技术鉴别市场上出售的牛肉及其制品的原料来源,用以判定制品是否为牛肉制品。分别提取牛肉、牛肉丸及牛肉粒三种样品的基因组DNA,以牛肉线粒体细胞色素氧化酶COⅠ基因设计引物进行PCR扩增,将PCR产物进行凝胶电泳分析,对三种样品PCR扩增条带进行分析,发现三种样品碱基数目相仿,可以推断为三种样品来源为同一物种,再结合引物特异性,可以推断三种样品均含有牛肉成分。     

外周血单个核细胞感染乙型肝炎病毒在母婴垂直传播中的作用

目的:探讨HBsAg、HBeAg阳性孕妇外周血单个核细胞(PBMC)内乙型肝炎病毒(HBV)DNA感染状况及其在宫内母婴垂直传播中的作用.方法:对HBsAg/HBeAg双阳性共67对孕妇及其新生儿静脉血分离和提纯PBMC后,经抽提、纯化后的DNA进入PCR扩增反应,引物为HBV C区基因序列.结果:67名HBsAg及HBeAg双阳性的孕妇中有35例(52.2%)PBMC中HBV DNA阳性,25例孕妇在血清及PBMC中均发现HBV DNA.67名新生儿有22例感染HBV DNA,感染率32.8%,其中血清HBV DNA阳性者10例,PBMC HBVDNA阳性者19例,二者均阳性者7例.结论:母亲PBMC内HBV DNA阳性可能导致新生儿PBMC中HBV DNA阳性,PBMC内的HBV DNA可能是HBV母婴垂直传播的一条重要途径,同时,HBsAg及HBeAg阳性母亲若血清HBV DNA为阳性就极大增加了其新生儿感染HBV的危险性.     

An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonuclease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5'-phosphorylated, circularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.     

A molecular biological study on identification of common septicemia bacteria using 16s–23s rRNA gene spacer regions

In the search for a rapid and reliable method for identification of bacteria in blood and cerebrospinal fluid , we developed a unified set of primers and used them under polymerase chain reaction(PCR) to amplify the spacer regions between the 16s and 23s genes in the prokaryotic rRNA genetic loci . Spacer regions within these loci showed a significant level of length and sequence polymorphism across most of the species lines. A generic pair of priming sequences was selected from highly conserved sequences in the 16s and 23s genes occurring adjacent to these polymorphic regions. This single set of primers and reaction conditions were used for the amplification of the 16s-23s spacer regions for 61 strains of standard bacteria and corresponding clinical isolates belonging to 20 genera and 27 species, including Listeria, Staphylococcus and Salmonella species, et al. When the spacer amplification products were resolved by electrophoresis, the resulting patterns could be used to distinguish most of the bacteria species within the test group, and the amplification products of the clinical isolates clustered at the standard species level. Some species presenting similar pattern were further analyzed by HinfI or AluI digestion or DNA clone and sequences analysis in order to establish the specific 16s-23s rRNA gene spacer regions map. Analysis of 42 blood specimens from septicemic neonates and 6 CSF specimens from suspected purulent meningitis patients by bacterial culture and PCR-RFLP(Restriction Fregament Length Polymorphism) showed that 15 specimens of blood culture were positive(35.7%) in the 42 septicemic neonates; 27 specimens were positive(64.2%) by PCR, and that the positive rate by PCR was significantly higher than that by blood culture(P<0.01). Among the 6 CSF specimens, one specimen found positive by blood culture was also positive by PCR, two found negative by blood culture showed positive by PCR; all three were S.epidermidis according to the DNA map. One C.neoformans found positive by blood culture showed negative by PCR. The remaining two specimens were both negative by PCR and blood culture. These results indicated that the method of detecting bacterial 16s-23s rRNA spacer regions using PCR and RFLP techniques was rapid, sensitive and specific in the detection of bacterial infections; and so, has very important application in the clinical diagnosis of sepsis in neonates.     

破伤风类毒素单克隆抗体的制备及初步鉴定

目的：制备并鉴定破伤风类毒素单克隆抗体杂交瘤细胞株,为破伤风的快速检测及致病机制的研究提供实验材料。方法：用破伤风类毒素做抗原免疫BALB/C小鼠,取其脾细胞与小鼠骨髓瘤细胞融合,获得分泌高滴度针对破伤风类毒素的杂交瘤细胞株,测定单抗免疫球蛋白亚类及单抗效价,用间接ELISA和Western-blot检测单克隆细胞株的特异性。结果：通过细胞融合和克隆化,筛选出3株持续分泌抗破伤风类毒素单克隆抗体的杂交瘤细胞株1E12、1E11、1G10。间接ELISA和Western-blot检测结果表明1E12、1E11、1G10可以和破伤风类毒素发生特异性反应。结论：成功制备了抗破伤风类毒素单克隆抗体,为制备免疫诊断试剂盒和抗体药物的开发奠定了基础。     

具有毒性质粒沙门菌荧光PCR快速检测试剂盒的研发

介绍了用于检测具有毒性质粒沙门菌的2种荧光PCR试剂盒（一种是荧光PCR染料法检测试剂盒,另一种是荧光PCR探针法检测试剂盒）的研制过程,2种试剂盒运用了自行设计的特异性引物和TaqMan探针,能同时特异性地从众多的沙门氏菌和非沙门氏菌中高效、快速、准确地检测出多种具有毒性质粒沙门氏菌,而对于不具有毒性质粒的沙门氏菌和非沙门氏菌均不能检出,可为食品检验和医疗诊断领域提供方便快捷检测具有毒素质粒沙门氏菌的荧光PCR试剂盒．     

单核细胞增生李斯特菌三种检测方法的比较

目的：通过比较国标法、real—timePcR法和LAMP方法,得到适合基层实验室快速检测单核细胞增生李斯特菌的方法。方法：分别用国标法、real-timePcR法和环介导的等温扩增（100p—mediated isothermal amplification,LAMP）法检测李斯特菌,并进行方法比较。结果：三种方法对单增李斯特菌的检测结果一致．国标法整个检测过程需5～7d;real—timePCR法实验条件要求高,成本高,检测需1．5～2．5d;LAMP法实验条件低,成本低,检测时限与real—timePCR相同。结论：LAMP~检测单增李斯特菌灵敏度高、特异性强、快速高效和低成本,适合基层实验室应急检测和现场监测使用。     

猪圆环病毒Ⅱ型河南地方株ORF4的克隆及原核表达

根据GenBank数据库中猪圆环病毒Ⅱ型的基因组序列设计引物,采用PCR技术从病料基因组DNA中扩增出PCVⅡ河南地方株ORF4基因,全长180 bp,编码59个氨基酸.将该基因克隆至载体pGEX-4T-3中形成pGEX-4T-3-ORF4表达载体.经PCR、酶切和测序鉴定后,转化表达菌株BL21（DE3）诱导表达.SDS-PAGE结果显示：ORF4能够在大肠杆菌中表达,产物的分子量约为32 kD,且以包涵体形式存在.Western Blot检测结果显示,纯化后的ORF4蛋白能够与鼠抗6＆#215;His标签单克隆抗体发生特异性反应,为进一步研究PCVⅡORF4蛋白的特性与功能奠定了基础.     

Detection of PCV2 DNA by SYBR Green I-based quantitative PCR

We developed an assay for the detection and quantitation of porcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 103 to 1011 copies of DNA per reaction. No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was de-tected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2.     

斑节对虾白斑综合症杆状病毒检测方法及其应用

对白斑和红体带白斑斑节对虾光镜和电镜观察，对虾白斑综合症杆状病毒（WSBV）感染外胚层和中胚层组织器官．应用WSBVPCR检测技术证实一种WSBV感染鳃、甲壳表皮、神经和肌肉，在肝胰腺、消化管（含胃）、心脏和血液中未检测到，其结果与组织学观察有出入．PCR检测WSBV最佳取材部位是对虾甲壳表皮和肌肉．PCR方法可应用于WSBV的提纯，实验用健康虾的筛选，实验感染WSBV的跟踪，以及流行病学研究．通过PCR方法和电镜观察结合证实红体病斑节对虾（不带白斑者）不是WSBV所致，修正了前人的观点．     

简并PCR法克隆L.starkeyi苹果酸酶基因

根据已报道的酵母苹果酸酶基因序列,利用CodeHop（Consensus Degenerate Hybrid Oligonucleotide Primers）软件设计两对简并引物,以斯达油脂酵母（L.starkeyi CICC 1809）总DNA为模板做简并PCR,得到2个基因片段（ME1,ME2）.对这2个目的片段进行测序,将结果翻译成氨基酸序列,blastp结果显示其中ME2片段为油脂酵母未报道苹果酸酶基因的序列（Gene Bank登录号GU348991）,并对巢式PCR方法检验简并PCR结果的有效性进行了评估.